Smad2 positively regulates the generation of Th17 cells - PubMed (original) (raw)

Smad2 positively regulates the generation of Th17 cells

Gustavo J Martinez et al. J Biol Chem. 2010.

Abstract

Development of Foxp3(+) regulatory T cells and pro-inflammatory Th17 cells from naive CD4(+) T cells requires transforming growth factor-β (TGF-β) signaling. Although Smad4 and Smad3 have been previously shown to regulate Treg cell induction by TGF-β, they are not required in the development of Th17 cells. Thus, how TGF-β regulates Th17 cell differentiation remains unclear. In this study, we found that TGF-β-induced Foxp3 expression was significantly reduced in the absence of Smad2. More importantly, Smad2 deficiency led to reduced Th17 differentiation in vitro and in vivo. In the experimental autoimmune encephalomyelitis model, Smad2 deficiency in T cells significantly ameliorated disease severity and reduced generation of Th17 cells. Furthermore, we found that Smad2 associated with retinoid acid receptor-related orphan receptor-γt (RORγt) and enhanced RORγt-induced Th17 cell generation. These results demonstrate that Smad2 positively regulates the generation of inflammatory Th17 cells.

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Figures

FIGURE 1.

FIGURE 1.

Foxp3 expression is regulated by Smad2. A, CD25+Foxp3+ cells in a CD4+ T cell gate were analyzed in the indicated tissues from Smad2fl/flCD4Cre− (Smad2 WT) or Smad2fl/flCD4Cre+ (Smad2 tKO) mice. A representative dot plot is shown for each group in each tissue (left panels), and the combined results for 10–15 mice in each group are indicated (right panel). p values were calculated using Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. LN, peripheral lymph nodes; MLN, mesenteric lymph nodes. B, CD4+CD25−CD62LhiCD44lo naive T cells from Smad2 WT mice were cultured in the presence or absence of different ratios of Smad2 KO or WT CD4+CD25+ natural regulatory T cells in triplicate wells with irradiated T cell-depleted splenocytes and stimulated with 1 μg/ml anti-CD3. Proliferation was assayed 72 h after treatment by adding [3H]thymidine to the culture for the last 8 h. A representative of three independent experiments is shown. C and D, FACS-sorted naive CD4+CD25−CD62LhiCD44lo T cells from Smad2fl/flCD4Cre− (Smad2 WT) or Smad2fl/flCD4Cre+ (Smad2 tKO) mice were stimulated with plate-bound anti-CD3 and anti-CD28 under iTreg conditions (TGF-β, IL-2, anti-IFN-γ, and anti-IL-4) for 4 days. Foxp3 expression was analyzed by intracellular staining (C). After differentiation, cells were restimulated with anti-CD3 for 4 h, and cDNA was prepared. Gene expression profile was analyzed by real-time RT-PCR (D). Data were normalized to a reference gene Actb. The lower expression level for each gene was referred as 1. A representative of three independent experiments is shown. Error bars in B and D indicate mean ± S.D.

FIGURE 2.

FIGURE 2.

T cells lacking Smad2 are defective in differentiation into Th17 cells in vitro. FACS-sorted naive CD4+CD25−CD62LhiCD44lo T cells from Smad2fl/flCD4Cre− (Smad2 WT) or Smad2fl/flCD4Cre+ (Smad2 tKO) mice were stimulated with plate-bound anti-CD3 and anti-CD28 under the indicated Th17 conditions for 4 days. A, cells were then restimulated with PMA/ionomycin in the presence of Golgi inhibitor for 4 h, and IL-17- and Foxp3-expressing cells were assessed by intracellular staining. B, after differentiation, cells were restimulated with anti-CD3 for 4 h, and cDNA was prepared. Gene expression profile was analyzed by real-time RT-PCR. Data were normalized to a reference gene Actb. The lower expression level for each gene was referred as 1. A representative example of four independent experiments is shown. Error bars indicate mean ± S.D. Rel. mRNA levels, relative mRNA levels.

FIGURE 3.

FIGURE 3.

Smad2 deficiency in T cells ameliorates EAE disease development. EAE was induced in Smad2fl/flCD4Cre− (Smad2 WT) or Smad2fl/flCD4Cre+ (Smad2 tKO) female mice. A, disease score (mean ± S.D.) measured after the second MOG immunization (see “Experimental Procedures”) combining three independent experiments (Smad2 WT, n = 10; Smad2 tKO, n = 13). p values were calculated using Student's t test comparing the disease score within each day. *, p < 0.05; **, p < 0.005. B and C, mononuclear cells infiltrating the central nervous system from the EAE mice were isolated on day 14 after the second immunization and restimulated with PMA, ionomycin, and GolgiStop for 6 h. CD4 and CD11b expression was determined (B), and IL-17- or IFN-γ-expressing cells were measured by intracellular staining on a CD4+ T cell gate (C). In B and C, a representative dot plot is shown, and the combined results (frequency or total cell number) for each group are indicated. p values were calculated using Student's t test. *, p < 0.05, **, p < 0.005. D, splenocytes from the above mice were stimulated with MOG peptide, and cytokine expression levels were measured by ELISA. Data shown are a representative example of three independent experiments with consistent results. p values were calculated using Student's t test. *, p < 0.05.

FIGURE 4.

FIGURE 4.

Smad2 directly binds to RORγt and synergizes in the generation of Th17 cells. A, HEK 293 T cells were transiently transfected with 6×Myc-tagged Smad3, 6×Myc-tagged Smad2, 2×Myc-tagged Smad4, FLAG-tagged RORγt, and/or His-tagged TGF-βRI T202D. After 48 h, lysates were prepared and immunoprecipitated with an anti-FLAG mAb (IP) followed by immunoblotting (IB) with anti-FLAG or anti-Myc (bottom two panels). The top three panels indicate Western blot of whole cell lysates (WCL). B and C, FACS-sorted naive OT-II CD4+ T cells were activated with OVA peptide-pulsed splenic APCs under neutral (anti-IL-4, anti-IFN-γ, and anti-TGF-β) conditions and co-infected with two bicistronic retroviruses (IRES-GFP or IRES-hCD2) expressing ROR_γ_t-GFP, Smad2_-2SD-hCD2_, and/or GFP or hCD2 vector controls. After 4 days, GFP+ hCD2+-infected cells were FACS-sorted. B, GFP+ hCD2+ cells were stimulated with PMA, ionomycin, and GolgiStop for 4 h, and IL-17-producing cells were determined by intracellular staining. C, GFP+ hCD2+ cells were restimulated for 4 h with anti-CD3, and mRNA expression of the indicated genes was analyzed by real-time RT-PCR. The data shown were normalized to expression of a reference gene Actb. The lowest expression of each gene was referred to as 1 and corresponds to naive T cells. The data represent at least three independent experiments with consistent results. Error bars indicate mean ± S.D. Rel. mRNA levels, relative mRNA levels.

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